261 INFLUENCE OF IN VITRO MATURATION ON EPIGENETIC MARKS AND GENE EXPRESSION IN BOVINE OOCYTES

Abstract

In cattle, in vitro maturation (IVM) of oocytes is an integral part of assisted reproduction technology. However, only 30% of in vitro matured bovine oocytes develop to the blastocyst stage after fertilization (compared with 60% for in vivo matured oocytes), indicating critical involvement of maturation conditions in the developmental competence of oocytes. Oocytes for IVF and intracytoplasmic sperm injection in humans are typically allowed to mature in vivo after superovulation because IVM is not considered to be a safe medical procedure. Several studies have shown that assisted reproduction technology involving prolonged in vitro culture of human and ruminant embryos can be associated with increased risk of fetal or placental abnormalities due to aberrant DNA methylation of imprinted and non-imprinted genes. Similarities between the bovine large offspring syndrome and imprinting-related human Beckwith–Wiedemann syndrome and the general similarity of bovine and human pre-implantation development make bovine oocyte maturation and embryonic development an increasingly accepted model of human development. Differentially methylated regions and imprinting control regions for the bovine paternally imprinted gene H19 and the maternally imprinted genes PEG3 and SNRPN were identified and characterised in this study. The DNA methylation profiles of bovine oocytes could be determined by bisulfite treatment of DNA from pools of 10 oocytes, but no significant differences were observed between IVM in TCM medium with 20% O₂, in SOF medium with 5% O₂, or after in vivo maturation. In contrast, quantitative PCR analysis of single oocyte preparations (n ≥ 8) revealed significant differences between these groups in the expression of the 3 genes. We then investigated the expression of genes involved in other critical processes in the developing oocyte and early embryo by quantitative PCR, including SLC2A8 (glucose transport), GDF9 (growth factor), PRDX1 (antioxidant and intercellular communication), DNMT1a/b (maintenance of methylation), and DNMT3a/b (de novo methylation). We also studied IGF2R, an imprinted gene implicated in large offspring syndrome. We observed significant differences in the expression of several of these genes. Interestingly, the expression of DNMT3a and DNMT3b was significantly higher in in vitro matured oocytes than in in vivo matured oocytes and could result in the above-mentioned aberrant methylation patterns established later in embryonic development.